F. Eyskens, MD, PhD 1,2

Expanding Newborn ScreeningFiction and Facts

1 UZA, 2 PCMA vzw, Antwerp, Belgium

F. Eyskens, MD, PhD 1,21 UZA, 2 PCMA vzw, Antwerp, Belgium

Ministry of Health of the Flemish Community

VCBMAPCMA vzw

40.000-50.000 newborns screened/lab/year

Neonatal Mass Screening in Flanders from 01/01/2012

40.000-50.000 newborns screened/lab/year

• Phenylketonuria/Hyperphenylalaninemia

• Congenital Hypothyroidism

• Congenital adrenal hyperplasia

• Biotinidase deficiency

• MCADD, MADD, MMA, PA, GA1, IVA, MSUD

Proposed selective screening

• Galactosemia

New pilot screening programs:

• Lysosomal Storage Diseases

• Duchenne Muscular Dystrophy

• Cystic Fibrosis IRT (stop: 2003)

Proposed mass screening program

Doctoral thesis university of Antwerp 1997

* Pilot study from 1 may 2002-30 april 2004,

implemented in Flanders

Screening Program from January 2007

IRT screening: Results

• Total number screened (1987-1999): 372.674

• CF patients found: 92 (prevalence: 1/4000)

• False positives: 2.445 (0.7-1.25%)

• False negatives: 8 (Delfia: 6)

• Sensitivity (%): 92

• Specificity (%): 99.3

• Positive predictive value: 3.6 %

• Negative predictive value: 99.99%

• Important health problem

• Asymptomatic stage in natural evolution

• Accepted treatment for patients

• Secondary prevention by genetic counseling and prenatal diagnosis

• Facilities for further diagnosis

• Suitable test available: simple, reasonably priced,

repeatable, sensitive, specific, acceptable

• Natural history of the disease is well known

• Screening should be a continuous process

CRITERIA for Screening Programs

(WHO: Wilson & Jungner)

Recycling activity of the vitamin biotin

Valine

Isoleucine

Methionine

Threonine

Odd Chain FFA

CholesterolGlucose

Pyruvate

Lactate

Leucine

3-methylcrotonylcoA

3-methylglutaconylcoA

AcetylcoA

Krebs Cycle

SuccinylcoA

MalonylcoA

Fatty acids

MethylmalonylcoA

PropionylcoA, C3

CC

ACC

PC

PCC

3-hydroxyisovaleric, C5-OH

Methylcitric acid

Propionylglycine

3-Hydroxy-n-valeric acid

Tiglylglycine, C5:1

Ketone bodies, C2

MULTIPLE CARBOXYLASE DEFICIENCY

Not for public use

Screening: BIOTINIDASE DEFICIENCY

Not for public use

Neonatal mass screening for

biotinidase deficiency in the

province of Antwerp.

1988-2013 (included in screening program of Flanders since 2007).

• False-positive rate (prematures):0.01%

• False-negative results: none reported

• sensitivity (%): 100

• specificity (%): 99.99

• positive predictive value (%): 39

• Prevalence of profound deficiency: 1:38,000

One Test-One Analyte - One Disease

One Test-Multiple Analytes - Several Diseases

NEONATAL SCREENING TODAY……

Neonatal mass screening by multiplex technology

FIA-tandem MS,

LC-MS-MS

FIA-tandem MS,

LC-MS-MS

Neonatal mass

screening by

multiplex technology

Newborn Screening in the era of multiplex technologies

Pediatrics 2006; 117: S296-S307.

OA FAO AA Hb other

IVA MCAD PKU HbSS CH

GA1 VLCAD MSUD HbS/b

Th

CAH

HMG LCHAD HCY HbS/

C

BIOT

MCD TFP CIT1 GALT

MMA CUD ASA HEAR

PA TYR1 CF

BKT MADD

OA FAO AA Hb other

IVA MCAD PKU HbSS CH

GA1 VLCAD MSUD HbS/

bTh

CAH

HMG LCHAD HCY HbS/

C

BIOT

MCD TFP GALT

MMA CUD? HEAR

PA TYR1 CF

BKT MADD

Comparison: Core panel USA-Flanders

MS/MS Broad screening, Brussels 1999-2004 versus

well-defined screening program, Antwerp 2002-2008

120,000-185,000 newborns screened

PKU 10 11 1/11,960 1/15,000

TYR 1 1 1 1/119,596 1/185,000

MCADD 8 13 1/14,949 1/14,000

MADD 1 1 1/119,500 1/185,000

MMA 2 3 1/59,798 1/55,000

GA 1 2 0 1/59,7983-

methylglutaconic1 1/119,596

holocarboxylase 1 1/185,000

Pediatrics 2006;118:448-456

CAVE: false-positives

• Provides energy in

the postabsorptive

and fasted state

• Important energy

source for the heart

• Important during

exercise in skeletal

muscle

S. Houten, R. Wanders. J Inherit Metab Dis (2010): 33: 469-477

Substrate specificity of the different

beta-oxidation enzymes

R. Wanders et al. J Inherit Metab Dis (2010); 33: 479-494

225 250 275 300 325 350 375 400 425 450 475 500m/z0

100

%

0

100

%

C2 carnitine

C3 carnitine

C16 carnitine

C8 carnitine

C10:1 carnitineC6 carnitine

Normal

Normal vs MCAD deficiency

MCAD deficiency

THE MOST COMMON MUTATION FOUND IN MCADD BEFORE SCREENING

Molecular genetic analysis: confirming diagnosis

High incidence of unexpected mutation frequencies found by neonatal screening:

Pennsylvania,USA

Sydney, Australia

B. Wilcken: ”It is not yet clear which patients

(MCADD “variants”) with disorders diagnosed by

such screening would have become symptomatic if

screening had not been performed”.

E.M.Maier, Germany, EMG 2009:

novel missense mutations and protein misfolding

Diagnosis of MCADD

• VLCAD deficiciency

– C14:2, C14:1, C14

(C14:1/C16)

• MCAD deficiciency– C6, C8, C10:1, C10

• LCHAD / MTP

deficiciency– C16:1OH, C16OH, C18:1OH,

C18OH

Acyl-CoA

CPT1

Acylcarnitineout

Acylcarnitinein

Acyl-CoA

CACT

CPT2

FAO

Plasma

Acylcarnitineout

Acylcarnitinein

Acyl-CoA

CACT

FAO

CPT2

m/z220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510

%

0

100

%

0

100

Parents of 85ES+ 260

218221 277261 274 456347459

482

Parents of 85ES+

Healthy

VLCAD deficientC14:1

426

Control

Patient

Used by Permission by S. Houten, Metabolics.be 2012

Revision of Screening Strategy

VLCAD deficiciency

• C14:1, C14:2, C14, C16

• C14:1/C16 Ratio

VLCAD deficiciency

• C14:1

• C14:1/C2 Ratio

• C14:2, C14, C16

• (C12, C12:1)

• C14:1/C16 Ratio

A functional enzyme activity assay is the only reliable

method to predict the clinical course in patients with

VLCADD detected by newborn screening: patients

showing a <10% residual enzyme activity are at risk to

develop clinical disease (*)

Enzymatic assay VLCAD

Fibroblasts/Lymphocytes

(* U. Spiekerkoetter, Duesseldorf, Germany, 2009)

R. Wanders et al. J Inherit Metab Dis (2010): 33; 479-494

Confirmation of diagnosis

• The diagnosis was confirmed by enzyme activity measurement in lymphocytes (AMC, Amsterdam, The Netherlands).

• The residual enzyme activity of VLCADD was 0.61, 0.24 and <0.17 nmol/min/mg protein, resp.

(controls: 1.84-4.80 nmol/min/mg protein;

10% enzyme activity = 0.66).

0

0.66

0.67

1.83

1.84

4.80

X

X

X

• MS/MS is screening

• The diagnosis is made by: Organic acid analysis by GC-MS

Amino acid analysis by liquid chromatography

Enzymatic tests in tissues

Molecular genetic analysis

• DO NOT SCREEN IF YOU ARE UNABLE TO

PERFORM FURTHER INVESTIGATIONS AND

FOLLOW-UP OF PATIENTS AND PARENTS

CONFIRMATION OF DIAGNOSIS & Follow-up

Confirmatory analysis: MS/MS is doing it by itself

Nat Med. 2012 Nov;18(11):1596

• Feasibility

• Control

• --

• Demand

• Need

Information

Epidemiology

Registries

Disease confirmation

Clinical utility

Treatment

Expert Centers

Politics/Ethics

Cost-effectiviness

Quality assurance

Technology

Training

EQUITY

SUSTAINABILITY

MCAD,PKU,CH

MSUDFAOOABiot LSD

Screening is a dynamic proces

newborn screening expert group of the American College of Medical Genetics

Scoring according to test availability

MPSI,II, IVA and VI

Pompe

SHOULD WE SCREEN FOR (TREATABLE) LSDs?

Data on file, Genzyme Corporation.

Infantile-Onset Pompe Disease

Head Lag

Hirschhorn R, et al. In: The Metabolic and Molecular Bases of Inherited Disease. 2000:3389-3420.

• Cardiomegaly– Chest x-ray

– Echocardiogram

• Cardiomyopathy– Echocardiogram

• Short PR interval

and large QRS

complex– Electrocardiogram

With permission from B. Byrne, MD

Infantile-Onset Pompe Disease

Cardiac Manifestations

Copyright ©2009 American Academy of Pediatrics

Chien, Y.-H. et al. Pediatrics 2009;124:e1116-e1125

FIGURE 4 Survival and motor outcomes for patients whose disease was detected by

newborn screening compared with those whose disease was diagnosed clinically

Screening improves outcome

Disease Progression: Severe

MPS I

10 months 12 months

22 months 34 months

39 months

MPS VI

case

Clinical

presentation

X-rays

Skull

Cervical spine

Hand

Pelvis

Current

status

Discussion

points

Future plans

Route MPS

diagnosis

short stature (122.4 cm)

underweight (BMI 17.7)

camptobrachydactyly

(claw hands)

bossing forehead

macrocephaly (head circumference 57 cm)

coarse facial features

protrusion of the eyes

full cheeks & thick lips

cervical kyphosis

underdeveloped secondary

sexual characteristics

hypertrophied mucousmembranes nose/mouth

hepatomegaly

elbow flexion contracture

bilateral limited abduction shoulder joint

thoracic kyphosis

adenoid hypertrophy

flexed-knees

shortened Achilles

tendon

severe OSA

severe restrictive and obstructive lung disease

(VC 39% pred. value)

glue ears & SNHL

total IQ

mild bilateral macular degenerative changes

dysostosis multiplex

SNHL: sensorineural hearing loss

OSA: obstructive sleep apnea

VC: vital capacity

mild valvular disease(thickening aortic, mitral)

bilateral limited abduction hips

motoric decline: e.g. reduced walking endurance

MPS VI

case

Clinical

presentation

X-rays

Skull

Cervical spine

Hand

Pelvis

Current

status

Discussion

points

Future plans

Route MPS

diagnosis

① Homozygous missense mutation in the ARSB gene

c.937C>G [p.P313A]

② Urine GAG analysis (DMB test)

24.5 mg GAG/mmol creatinine (reference values 0.6–2.6)

③ Separation of the GAG by electrophoresis

presence of dermatan sulphate

absence of heparan sulphate

④ Leucocyte arylsulfatase B (ASB) activity

<0.5 nmol/mg/min (reference values 2.2–18.6)

Presented at Department of Radiology with knee

trauma

-- > conclusion:

no fracture

suspicion of skeletal dysplasia

= indication for a genetic referral

Age at diagnosis: 20 years!

Short Stature (MPS VI)

Oussoren E, Brands MM, Ruiter GJ et al. Biochim Biophys Acta 2011;812(11):1542-56.

The growth chart of a patient with MPS VI (open circles). Enzyme-

replacement therapy was started at 7 years of age (arrow).

Morquio A: Musculoskeletal manifestations

Harmatz et al, Mol Genet Metab, 2013; Dhawale et al, J Pediatr Orthop, 2012; White, Curr Orthop Prac, 2012 ;

Dhawale et al, J Pediatr Orthop B, 2012; Tomatsu et al, Curr Pharm Biotechnol, 2011

Skeletal dysplasia

– Spinal abnormalities

– Pectus carinatum

– Hip dysplasia

– Genu valgum

– Ankle valgus

– Hand abnormalities

– Flat facial features

– Mandibular protrusion

Left image: Kalteis et al, Arthroscopy, 2005

Right images: Atinga et al, J Bone Joint Surg Br, 2008

Short stature

Joint subluxation

Joint instability

Joint degeneration

Abnormal gait

Weak hand grip

Management includes regular imaging studies and

corrective surgery, physical therapy, pain management as needed, and

walker/wheelchair use

LSD incidence:

Current view of LSD incidence underestimated:

• Incidence of Fabry in Italy: 1/3100 births (Spada et al, 2006, Am J Hum Genet)

• Incidence of Fabry in Taiwan: 1/1250 (Lin et al, 2010, J Inherit Metab Dis)

• Incidence of Pompe in Taiwan: 1/41000 (Chien et al, 2009, Pediatrics)

• Incidence of 1 per 2315 births (3 LSD) (Mechtler et al, 2012, Lancet)

• Gaucher: 1/17000

• Pompe: 1/8700

• Fabry: 1/3900

• Incidence of Fabry, Pompe, and MPS-I is estimated at 1/7500 births (3 LSD) (Scott et al, 2013, J Pediatr)

• Fabry: 1/7800

• Pompe: 1/27800

• MPS-I: 1/35500

• Chamoles method (2001):

– Fluorescence/enzymatic assay

– Single assay-Single disease model

• Meikle et al (Hopwood)(2004-2006):

– Multiplexed immune quantification

– Specific antibodies-two-tier approach

– Low sensitivity for detection of Pompe & Gaucher

• Gelb/Li et al (2004); Genzyme (Zhang et al)(2008)

– ESI-MS/MS

– Analytically multiplex screening

– MPSI,II,VI, Pompe, Fabry, Gaucher, Niemann-Pick, Krabbe: specific substrates and Internal Standards

– ExtQC-CDC

• Millington

– Digital microfluidics platform

– =multiplex platform of Chamoles method

– MPSI, VII, Pompe, Fabry

Available Techniques

NBS for LSD worldwide:

• USA (Spacil 2013 et al, Clin Chem)

• Washington: pilot for Pompe, Fabry, Gaucher, Krabbe, Niemann-Pick A/B, MPS-I, MPS-II, MPS-IVA and MPS-VI

• New York – Krabbe screening

• several states passed legislation to include LSD screening

• Taiwan – Pompe screening (Chien et al, 2009, Pediatrics)

• Austria – Vienna: anonymous study for Gaucher, Pompe, Fabry and Niemann-Pick A/B (Mechtler et al, 2012, Lancet)

Method Fluorescence

Enzymatic assay

ESI-MS/MS

Enzymatic assay

Population Taiwan (2005-2008) Austria (2008)

N 132,538 10,279

Recall rate % 0.82 0.039

False

positives

117 4

False Positives: e.g. Pompe

Technological milestones:

• Mechtler et al. (J of Chromatogr B, 2011)

• Abolish liquid-liquid and solid phase extraction

• Short incubation (3h)

• Simultaneous quantification of five LSDs

• DBS punch and buffer 1: Gaucher, Niemann-Pick A/B, Pompe and Fabry diseases

• DBS punch and buffer 2: MPS-I

• Turboflow – UHPLC-MS/MS (no solid phase extraction and substrate interference)

• Cost (without MPS-I) = 1 euro/sample

Disadvantages for PCMA

• Some LSDs of interest are not included

• 2 DBS and buffers required

How to proceed:

• According to protocol Spacil et al. (Clin Chem, 2013):

• Assay of 7 enzymes in 1 or 2 buffers, with 1 or 2 punches:

• 4+3 plex assay: 2 DBS punches

• 1 for Pompe, Fabry, Gaucher, MPS-I in 4-plex buffer

• 1 for MPS-II, MPS-IVA, MPS-VI in 3-plex buffer

• 7 plex assay: all the above on 1 DBS punch in 1 assay possible with MS/MS (XEVO)

• Pre-analytical steps in 96 well plates

• Assay cocktail added to DBS

• 16h incubation

• Acetonitril added to quench reaction and precipitate proteins

• Centrifugation

• Supernatant transferred to other 96 well plate for analysis

• Injection on dual column (UPLC)

• MS/MS (Xevo) quantification of 7 enzyme product and internal standards

Dajnoki et al. Clin Chem 2008

Pompe Screening by MS/MS

Discrimination: infantile versus late-onset

Validation tests:

• Quality Control Assessment - Centers for Disease Control (CDC), Atlanta (De Jesus 2009 et al, Clin Chem)

Instrument calibration curve

Linearity

Precision and accuracy (CV)

Detection limit

Carry-over and column contamination

Evaluation of incubation time

Evaluation 4+3-plex compared to 7-plex

• Case study

• Stability study

• (Establish reliable reference range)

• 132,538 newborns screened

1:1239 genotyped: 2/3 pathogenic mutations;

1/3 pseudodeficiency

Good differentiation by lymphocyte enzymatic GAA

activity: low false positive rate

However: no discrimination between infantile and

late-onset disease!!

• Two-tier screening strategy is indicated, WHAT?

Pompe

Labrousse P et al: Mol Genet Metab 2010

Scala I et al: J Matern Fetal Neonatal Medicin

2012 Oct; 25 (Suppl 5): 4-6

• In the near future it will be also possible to

perform a genetic and mutational scan across

the whole genome of the fetus in a non-invasive

manner by analyzing cell-free fetal DNA in

maternal blood as early as the 5th week of

gestational age. These high-throughput methods

applied to neonatal and non-invasive prenatal

screening of genetic diseases, including inborn

errors of metabolism, are raising further

technical, political and ethical issues.

Ethics?

It is not about how we will screen, but

What and Why should we screen?

• Mass screening of newborns should stay centralised in

those screening labs that have the most experience and

the best performance; only these labs can implement new

techniques and new screening programs

• Multiplex technology MS/MS have changed screening

strategies

• Neonatal mass screening programs:

– differ depending on the population screened

– are not determined by the available technology

• Screening is a dynamic rather than a static proces

CONCLUSIONS

• PCMA vzw

• Local Government of the province of Antwerp

• Minister of Health of the Flemish Community and Administration

• All colleagues from Belgium, The Netherlands, Luxembourg,

Austria, USA (CDC, Washington), SGS-Ewacs

• PerkinElmer, Waters, Biomarin, Shire, Genzyme

THANK YOU